Profilin is a ubiquitous 15kD protein required for the normal distribution of filamentous actin structures in vivo. Profilin binds PIP2, suggesting a possible link to signal transduction pathways. Profilins from different organisms have limited sequence homology, but highly homologous 3D structures, indicating an evolutionary pressure to maintain the protein fold. It has been the goal of our laboratory to combine crystallography and in vivo studies to examine the role of profilin in the cell. Two profilin data sets were collected at X9B. Crystals of P2 profilin, (a profilin isoform of Acanthamoeba castellani), which diffracted only to 4.0A using a rotating anode and diffracted to 2.0A at X9B, has a space group of F432 and unit cell parameters of a=b=c=241.28, a=b=g=90o. The atomic structure of P2 profilin will aid in the creation of mutants with which to study the specific lipid binding activity of this protein. Crystals of Saccharomyces cerevisiae profilin which diffracted weakly using a rotating anode, diffracted to 3.0 A at X9B. The S. Cerevisiae crystals are of the P432 space group with unit cell parameters of a=b=c=127.66, a=b=g=90o and were successfully reduced and scaled (Rmerge of 5.8%). We have now prepared a new crystal form of yeast profilin (P65; a=b=58.1, c=151.3E) and have refined the structure to 2.3E resolution. This structure is being used to design specific mutants that will probe the in vivo role of actin and proline rich-ligand binding. Furthermore, we have recently solved the structure of human profilin bound to a decamer of L-proline which gives the first structural information about this interaction that is responsible for the intra-cellular localization of profilin.